CDK5 抑制通过调控 Drp1S616 磷酸化防止 OGDR 诱导的线粒体碎片化和细胞凋亡。
CDK5 inhibition protects against OGDR induced mitochondrial fragmentation and apoptosis through regulation of Drp1S616 phosphorylation.
机构信息
Department of Neurology, Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, China.
Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan 410078, China; Hunan Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan 410078, China; Hunan Key Laboratory of Animal Model for Human Diseases, Central South University, Changsha, Hunan 410078, China.
出版信息
Life Sci. 2021 Mar 15;269:119062. doi: 10.1016/j.lfs.2021.119062. Epub 2021 Jan 18.
AIMS
Cyclin-dependent kinase 5 (CDK5) is a potential target for the treatment of cerebral ischemia. CDK5 is one of the upstream regulators for Dynamin-related protein 1 (Drp1) phosphorylation. This study intends to discuss whether CDK5 inhibition conferring neuroprotection in cerebral ischemia through regulating Drp1 phosphorylation.
MATERIALS AND METHODS
Mouse neuroblastoma N2a cells and N1E-115 cells were cultured and subjected to oxygen-glucose deprivation/reperfusion (OGDR). N2a cells and N1E-115 cells were treated with Roscovitine, a pharmacological inhibitor of CDK5, or transfected with CDK5 siRNA to knock down CDK5 expression. N2a cells were transfected with different plasmids (Drp1-Myc, the dephosphorylation-mimic mutant Drp1S616A-Myc and the phosphorylation-mimic mutant Drp1S616D-Myc). The expression of CDK5 and its activator p35, Drp1 and phosphorylated Drp1 on S616 was determined by western blot. The morphology of mitochondria was detected by immunofluorescence staining and the proportion of N2a cells with apoptosis was detected by flow cytometry analysis.
KEY FINDINGS
Expression of CDK5, p35 and phosphorylated Drp1 on S616 was strongly upregulated after 4 h and 12 h reperfusion following 4 h oxygen-glucose deprivation (OGD) at protein level. CDK5 inhibition by pre-treated with Roscovitine or transfection with CDK5 siRNA significantly ameliorated OGDR induced mitochondrial fragmentation and apoptosis. Overexpression of the phosphorylation-mimic mutant Drp1S616D abrogated the protective effect of CDK5 inhibition against OGDR induced mitochondrial fragmentation and apoptosis.
SIGNIFICANCE
Our data indicate that the neuroprotective effect of CDK5 inhibition against OGDR induced neuronal damage is Drp1S616 phosphorylation dependent. A better understanding of the neuroprotective mechanisms of CDK5 inhibition in cerebral ischemia will help to develop safe and efficacious drugs targeting CDK5 signaling for clinical use.
目的
细胞周期蛋白依赖性激酶 5（CDK5）是治疗脑缺血的潜在靶点。CDK5 是动力相关蛋白 1（Drp1）磷酸化的上游调节因子之一。本研究旨在探讨 CDK5 抑制是否通过调节 Drp1 磷酸化来发挥脑缺血的神经保护作用。
材料和方法
培养小鼠神经母细胞瘤 N2a 细胞和 N1E-115 细胞，并进行氧葡萄糖剥夺/再灌注（OGDR）。用 Roscovitine（CDK5 的药理学抑制剂）处理 N2a 细胞和 N1E-115 细胞，或用 CDK5 siRNA 转染以敲低 CDK5 表达。用不同的质粒（Drp1-Myc、去磷酸化模拟突变体 Drp1S616A-Myc 和磷酸化模拟突变体 Drp1S616D-Myc）转染 N2a 细胞。通过 Western blot 测定 CDK5 及其激活剂 p35、Drp1 和 S616 磷酸化 Drp1 的表达。通过免疫荧光染色检测线粒体形态，通过流式细胞术分析检测 N2a 细胞凋亡的比例。
主要发现
在 4 小时氧葡萄糖剥夺（OGD）后再灌注 4 小时和 12 小时时，CDK5、p35 和 S616 磷酸化 Drp1 的表达在蛋白质水平上均强烈上调。用 Roscovitine 预处理或用 CDK5 siRNA 转染预先抑制 CDK5 可显著改善 OGDR 诱导的线粒体片段化和凋亡。过表达磷酸化模拟突变体 Drp1S616D 可消除 CDK5 抑制对 OGDR 诱导的线粒体片段化和凋亡的保护作用。
意义
我们的数据表明，CDK5 抑制对 OGDR 诱导的神经元损伤的神经保护作用依赖于 Drp1S616 磷酸化。深入了解 CDK5 抑制在脑缺血中的神经保护机制将有助于开发针对 CDK5 信号的安全有效的药物，用于临床应用。
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